This project constitutes an investigation into mechanisms whereby cells achieve the homeostasis of calcium ion concentration, within the cytosol and other cell compartments, and allow perturbations in calcium in response to hormones and neurotransmitters. This year we have investigated the magnitude of the magnesium ion gradient across the mitochondrial membrane and its regulation. This is part of our ongoing study of the regulation of dehydrogenase activity by calcium ions as the pyruvate and 2-oxoglutarate dehydrogenases are activated by intramitochondrial calcium, with a sensitivity that depends upon the magnesium ion concentration. Intramitochondrial free magnesium was measured by use of the newly-available fluorescent chelating agent Mag-fura, and a dual-excitation spectrofluorimeter. It was found to be in the range of 0.3 to 2 mM, depending on the presence of extramitochondrial magnesium and adenine nucleotides: - both ATP and ADP increased mitochondrial free magnesium. Polyamines, e.g., spermine, also increased magnesium, in a process which was dependent on extramitochondrial free calcium. In addition, exposure of mitochondria calcium ion concentrations which are plausible for activated cells (0.5 to 1 micromolar) gave rise to large increases in free intramitochondrial magnesium: this may form part of the mechanism whereby elevated cytosolic calcium raises the fraction of active, dephospho pyruvate dehydrogenase in the mitochondria.